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J. Biol. Chem., Vol. 280, Issue 3, 1826-1837, January 21, 2005

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ADAM10 Mediates Ectodomain Shedding of the Betacellulin Precursor Activated by p-Aminophenylmercuric Acetate and Extracellular Calcium Influx^*

Michael P. Sanderson, Sarah N. Erickson, Peter J. Gough¶, Kyle J. Garton¶, Paul T. Wille¶, Elaine W. Raines¶, Andrew J. Dunbar||**, and Peter J. Dempsey

From the Pacific Northwest Research Institute, Seattle, Washington 98122, the Cooperative Research Centre for Tissue Growth and Repair, School of Biological Sciences, Flinders University, Adelaide 5001, Australia, the ^¶Department of Pathology, University of Washington, Harborview Medical Center, Seattle, Washington 98104, ^||GroPep Limited, Thebarton, South Australia 5031, Australia, and the Department of Medicine, University of Washington, Seattle, Washington 98195

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26–28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase C inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.

Received for publication, August 2, 2004 , and in revised form, October 1, 2004.

^* This work was supported in part by the Australian Government Cooperative Research Centres Programme (to A. J. D.) and National Institutes of Health Grants DK59778 and DK63363 (to P. J. D.) and Grants HL18645 and HL67267 (to E. W. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence may be addressed: GroPep Ltd., 28 Dalgleish St., Thebarton, SA 5031, Australia. Tel.: 61-8-8354-7797; Fax: 61-8-8354-7788; E-mail: andrew.dunbar{at}gropep.com.au. To whom correspondence may be addressed: Pacific Northwest Research Inst., 720 Broadway, Seattle, WA 98122. Tel.: 206-568-1462; Fax: 206-726-1217; E-mail: pdempsey{at}pnri.org.

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